Gonorrhea, which is caused by Neisseria gonorrhoeae, is a sexually transmitted disease that currently is categorized by the Centers for Disease Control and Prevention as one of the four ?urgent level? drug-resistant threats to the United States. The disease is prevalent in active Military and rates appear to be elevated in Veteran populations. Although quinolones were used routinely to treat gonorrhea starting in 1993, their use as front-line therapy was discontinued in 2006 due to the high incidence of resistance. The cellular targets of quinolones are the bacterial type II topoisomerases, gyrase and topoisomerase IV. The identification and characterization of novel agents that act against these well-validated enzyme targets, but overcome the associated resistance, could have important ramifications for the clinical treatment of gonorrhea. Gyrase and topoisomerase IV are essential enzymes that regulate DNA under- and overwinding and remove DNA knots and tangles by generating transient double-stranded breaks in the genetic material. Quinolones kill bacteria by increasing the levels of these gyrase- and topoisomerase IV-generated double- stranded DNA breaks, which converts these enzymes into lethal proteins that fragment the genome. Both enzymes are targets for quinolones, but their importance to drug action is species- and drug-dependent. Initial quinolone resistance is most often associated with specific mutations in gyrase and/or topoisomerase IV that occur at a highly conserved Ser residue or a Glu/Asp located 4 residues downstream. Based on a published structure and a series of functional studies from the Osheroff laboratory that delineated interactions between drugs and the enzymes from Bacillus anthracis, Escherichia coli, and Mycobacterium tuberculosis, these residues anchor a water-metal ion bridge that serves as the primary conduit between quinolones and the type II enzymes. By characterizing quinolone-topoisomerase interactions, the PI has designed novel drugs that overcome resistance due to mutations in M. tuberculosis gyrase and B. anthracis gyrase and topoisomerase IV. Recently, a new class of naphthyridone/aminopiperidine-based agents, ?novel bacterial topoisomerase inhibitors? (NBTIs), was reported. NBTIs target bacterial type II topoisomerases but display little or no cross- resistance to clinically relevant quinolone resistance mutations in gyrase or topoisomerase IV. Unlike the quinolones, these agents either act as catalytic inhibitors or induce enzyme-mediated single-stranded DNA breaks. However, no additional mechanistic information has been reported for any member of this drug class. Gepotidacin, an NBTI that is in clinical trials against gonorrhea, displays activity against wild-type and quinolone-resistant N. gonorrhoeae cultures. However, neither its actions, nor those of any other NBTI against N. gonorrhoeae gyrase or topoisomerase IV have been described. There is an urgent need to develop new drugs to treat resistant gonorrhea (as well as other resistant bacterial infections). The premise that underlies the proposed research is that understanding how drugs interact with their enzyme target places us in a far better position to develop drugs that overcome resistance. Thus, the specific aims of this proposal are to 1) determine the mechanistic basis for quinolone action against N. gonorrhoeae gyrase and topoisomerase IV, define the basis for target-mediated quinolone resistance, and utilize the findings to identify quinolones that overcome the most common forms of resistance; and 2) determine the mechanistic basis for the actions of NBTIs against N. gonorrhoeae gyrase and topoisomerase IV. Although the primary research models for this study will be N. gonorrhoeae gyrase and topoisomerase IV, cellular studies also are planned. In addition, some of the proposed studies may utilize M. tuberculosis, E. coli, or B. anthracis models for comparative purposes. Finally, the proposed research benefits greatly from previous studies from the Osheroff laboratory on the mechanism of bacterial and eukaryotic type II topoisomerases and the interaction of these enzymes with quinolones and other drugs.